豬環狀病毒第二型Cap蛋白之表現及結構分析

Porcine circovirus type 2 (PCV2) is the primary causative agent of the economically important global disease, porcine circovirus associated diseases (PCVAD). The viral capsid protein (Cap) containing the major antigenic sites has become the target protein for developing of diagnostic reagents and PCV2 subunit vaccine. The purpose of this study is to express a variety of recombinant Cap proteins using an E. coli expression system for further analysis of the structure and immunogenicity of viral capsid. Four recombinant Cap proteins including the rCapT7+ containing a N-terminal T7 tag, the rCapHis+ containing a C-terminal His tag, the rCapT7+His+ containing both tags at each end, and the rCap containing no tag were constructed. The recombinant Cap proteins were purified by the continuous sucrose gradient ultracentrifugation and confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. All the expressed Cap proteins no matter containing a fusion tag could self-assemble into virus-like particles (VLPs) by the transmission electronmicroscope analysis. The immunogenicity of PCV2 VLPs were further evaluated in mice. Both the rCap and fusion tags containing rCapT7+His+ VLPs immunized groups were able to elicit specific antibody responses to PCV2 with neutralizing antibody titers of 1:8 after twice booster immunization. In addition, both immunized groups showed detectable of IFN- secreting cells response in the ELISpot analysis. All the results demonstrated that the PCV2 VLPs based on the E. coli-expressed Cap proteins could mount both humoral and cellular immune response against PCV2 Cap in mice, and the terminal fusion tags had no significant effect on structural assembly and immunogenicity of Cap protein.摘要 i Abstract ii 目次 iii 表目次 v 圖目次 vi 第一章 前言 1 第二章 文獻探討 3 一、豬環狀病毒的簡介 3 二、豬環狀病毒的特性 4 三、豬環狀病毒的複製與基因表現 4 四、病毒外殼蛋白簡介 7 五、 豬環狀病毒的致病機轉 8 六、 PCV2免疫調節作用 10 七、 豬環狀病毒的診斷和預防 11 八、 PCV2疫苗的發展 12 九、 單株抗體簡介 14 第三章 材料與方法 16 1. PCV2密碼子優化之ORF2重組表現質體之構築 16 1.1. PCV2 rORF2、rORF2T7+ 及rORF2His+ 基因序列的選殖 16 1.2. 純化DNA片段 16 1.3. 重組表現質體之構築 16 1.4. 大腸桿菌轉型作用 (Transformation) 17 2. PCV2 Cap重組蛋白之表現及特性分析 17 2.1. 利用大腸桿菌表現Cap重組蛋白 17 2.2. 蛋白質電泳 (Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, SDS-PAGE) 18 2.3. 西方墨點法 (Western blotting) 18 3. PCV2類病毒顆粒組裝之研究 19 3.1. 連續性蔗糖濃度梯度超高速離心純化VLP 19 3.2. 穿透式電子顯微鏡 (Transmission electron microscopy, TEM) 19 3.3. 免疫金粒子標識測定分析 (Immunogold labelling) 19 4. 製備抗PCV2 Cap之特異性單株抗體 20 4.1. 小鼠免疫試驗 20 4.2. 融合瘤試驗 (Hybridoma experiment) 20 4.3. 棋盤式力價測定法 (Box titration) 21 4.4. 間接螢光抗體試驗 (Indirect immunofluorescence assay, IIF) 22 4.5. 融合瘤的篩選 22 4.6. 融合瘤細胞之單株化 (Cloning) 22 4.7. 生產腹水 23 4.8. 抗體亞型鑑定 (Isotyping) 23 4.9. 抗原決定位分析 (Epitope mapping) 23 5. PCV2類病毒顆粒之抗原性分析 24 5.1. 小鼠免疫試驗 24 5.2. 血清學檢測 24 5.3. 利用ELISpot (Enzyme-linked immunospot) 偵測IFN--SC的反應 24 5.4. 血清中和試驗 (Serum neutralization test) 25 第四章 結果 27 1. 利用大腸桿菌表現系統表現PCV2重組外殼蛋白 27 1.1. 表現rCapT7+His+ 重組蛋白 27 1.2. 免疫金粒子標識分析PCV2 rCap T7+His+ 重組蛋白 27 1.3. PCV2 Cap重組蛋白表現及特性分析 27 2. PCV2類病毒顆粒組裝之研究 28 2.1. Cap重組蛋白之純化及特性分析 28 2.2. 全長Cap重組蛋白可自行組裝形成VLP 28 3. 製備抗PCV2 Cap之特異性單株抗體 29 3.1. 融合瘤實驗與融合瘤細胞的篩選及單株化 29 3.2. 單株抗體之特性分析 29 4. PCV2類病毒顆粒之免疫原性分析 30 4.1. 重組蛋白rCap及rCapT7+His+ 引起小鼠特異性抗體免疫反應 30 4.2. 重組蛋白rCap及rCapT7+His+ 引起小鼠細胞性免疫反應 30 第五章 討論 49 參考文獻 5